Abstract Rapid and accurate fever diagnosis represents a challenge to health clinics serving resource-poor settings where fevers caused by specific infectious diseases require immediate and disease-specific treatment or quarantine response. Development of laboratory-quality tests that can be applied at the point of care requires simplification of assay protocols and development of compact, rugged, and quantitatively accurate instrumentation capable of reading/reporting assay results. In the proposed project, we combine complementary innovation in molecular biology methods, spectroscopy, and instrumentation to develop the core of a system that can perform multiplexed, rapid, and highly sensitive analysis of infectious disease biomarkers in a single droplet of serum. First, we utilize a novel assay format to enhance existing laboratory- based Enzyme Linked Immunosorbent Assays (ELISA). In this method, incorporation of a cleavable dsDNA linker on the detection antibody enables it to be co-immobilized with its complementary capture antibody. This process allows for a rapid and streamlined assay protocol with a single cooperative antigen binding step and improved sensitivity compared to conventional ELISAs. Second, we develop a nanostructured optical transmission filter component that, when attached to a conventional photodetector array imaging chip, converts it into a spectrometer capable of measuring the absorption spectra within 10 microfluidic channels in parallel. The assay protocol is implemented within a transparent microfluidic cartridge that is placed into a low-cost handheld detection instrument that integrates the spectrometer, LED illumination, and a single syringe controlled by a linear actuator that drives the entire assay protocol using pneumatic force. Third, our instrument communicates by a wireless Bluetooth interface with a smartphone, which runs a software application for control of the instrument, data processing, and data communication with a smart service system. The resulting detection complex, microfluidic assay protocol, absorption spectrometer, instrument, and smartphone application represents a broadly applicable and substantially simplified method for providing rapid lab grade point-of-care diagnostics to health care providers.